北京棒杆菌单体天冬氨酸激酶T379N/A380C/T65I/D173G突变体酶学性质表征及工程菌构建

Q517; 选取天冬氨酸激酶抑制剂赖氨酸(Lys)的结合位点苏氨酸(Thr) 379和丙氨酸(Ala) 380、底物天冬氨酸(Asp)的结合位点Thr65、既是底物也是ATP结合位点的催化活性中心Asp 173,对4个关键残基位点进行定点饱和突变.采用高通量筛选方法成功构建突变体T379N/A380C/T65I/D 173G,与野生型(wild type,WT)相比,酶活力提高到75.83倍.动力学与酶学性质表征显示:相比于WT,Km值由4.11 mmol/L降低至1.34 mmol/L,表明与底物亲和力增强;n值由1.71减小到1.07,正协同性降低.突变体T379N/A380C/T65I/...

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Published in食品科学 Vol. 43; no. 2; pp. 100 - 107
Main Authors 王亚南, 刘晓婷, 樊占青, 王哲人, 高欣, 闵伟红
Format Magazine Article
LanguageChinese
Published 吉林农业大学食品科学与工程学院,小麦和玉米深加工国家工程实验室,吉林长春 130118 25.01.2022
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北京棒杆菌;天冬氨酸激酶;突变体;酶学性质;工程菌构建
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ISSN1002-6630
DOI10.7506/spkx1002-6630-20201110-103

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Abstract Q517; 选取天冬氨酸激酶抑制剂赖氨酸(Lys)的结合位点苏氨酸(Thr) 379和丙氨酸(Ala) 380、底物天冬氨酸(Asp)的结合位点Thr65、既是底物也是ATP结合位点的催化活性中心Asp 173,对4个关键残基位点进行定点饱和突变.采用高通量筛选方法成功构建突变体T379N/A380C/T65I/D 173G,与野生型(wild type,WT)相比,酶活力提高到75.83倍.动力学与酶学性质表征显示:相比于WT,Km值由4.11 mmol/L降低至1.34 mmol/L,表明与底物亲和力增强;n值由1.71减小到1.07,正协同性降低.突变体T379N/A380C/T65I/D 173G最适反应温度由WT的25℃提高到30℃,最适pH值与WT相同均为8.0,半衰期由WT的4.24 h缩短至3.23 h.突变体在0.2～10 mmol/L抑制剂浓度下,抑制作用整体减弱,尤其在Lys和Lys+Thr作用下明显激活.通过无缝克隆技术电转入北京棒杆菌构建工程菌,发酵测得Lys产量相比WT提高83.05％,Thr提高29.36％,甲硫氨酸(Met)提高30.77％,为优化天冬氨酸激酶代谢途径和构建高产天冬氨酸族氨基酸菌株提供参考.
AbstractList Q517; 选取天冬氨酸激酶抑制剂赖氨酸(Lys)的结合位点苏氨酸(Thr) 379和丙氨酸(Ala) 380、底物天冬氨酸(Asp)的结合位点Thr65、既是底物也是ATP结合位点的催化活性中心Asp 173,对4个关键残基位点进行定点饱和突变.采用高通量筛选方法成功构建突变体T379N/A380C/T65I/D 173G,与野生型(wild type,WT)相比,酶活力提高到75.83倍.动力学与酶学性质表征显示:相比于WT,Km值由4.11 mmol/L降低至1.34 mmol/L,表明与底物亲和力增强;n值由1.71减小到1.07,正协同性降低.突变体T379N/A380C/T65I/D 173G最适反应温度由WT的25℃提高到30℃,最适pH值与WT相同均为8.0,半衰期由WT的4.24 h缩短至3.23 h.突变体在0.2～10 mmol/L抑制剂浓度下,抑制作用整体减弱,尤其在Lys和Lys+Thr作用下明显激活.通过无缝克隆技术电转入北京棒杆菌构建工程菌,发酵测得Lys产量相比WT提高83.05％,Thr提高29.36％,甲硫氨酸(Met)提高30.77％,为优化天冬氨酸激酶代谢途径和构建高产天冬氨酸族氨基酸菌株提供参考.
Author 高欣
王亚南
樊占青
刘晓婷
闵伟红
王哲人
AuthorAffiliation 吉林农业大学食品科学与工程学院,小麦和玉米深加工国家工程实验室,吉林长春 130118
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FAN Zhanqing
WANG Yanan
GAO Xin
LIU Xiaoting
MIN Weihong
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DocumentTitle_FL Enzymatic Characterization and Engineering of Monomer Aspartokinase Mutant T379N/A380C/T65I/D 173G from Corynebacterium pekinense
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Keywords 北京棒杆菌;天冬氨酸激酶;突变体;酶学性质;工程菌构建
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Snippet Q517; 选取天冬氨酸激酶抑制剂赖氨酸(Lys)的结合位点苏氨酸(Thr) 379和丙氨酸(Ala) 380、底物天冬氨酸(Asp)的结合位点Thr65、既是底物也是ATP结合位点的催化活性中心Asp...
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Title 北京棒杆菌单体天冬氨酸激酶T379N/A380C/T65I/D173G突变体酶学性质表征及工程菌构建
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